Acute myelogenous leukemia (AML) is the deadliest and most common form of adult leukemia. Each year, over 10,000 people in the United States are diagnosed with AML. While most patients experience a remission of the cancer after initial chemotherapy treatments, approximately 80% subsequently relapse. Fewer than 30% of AML patients survive past one year after relapsing. The high rate of AML relapse has been attributed to a small fraction of cells termed leukemia stem cells. LSCs are highly resistant to multiple chemotherapeutic drugs, and survive initial chemotherapy treatments. They are capable of aggressively restoring the leukemia cell population. Developing a method to target and kill LSCs would benefit AML patients tremendously. However, progress in studying LSC biology has been limited because of the low numbers that can be isolated from patients. This research will develop technology that can investigate, on the single cell level; the sphingosine kinase (SphK) activity of LSCs. SphK is an oncogenic kinase that is known to increase resistance to chemotherapeutic drugs in leukemia cells. The central hypothesis of this proposal is that SphK is up regulated in LSCs, which would make it a promising target for drug therapy. SphK activity will be measured by loading cells with a fluorescent reporter, which can be modified by active SphK. Single-cell capillary electrophoresis (CE) will be used to separate the modified from the unmodified reporter in each cell. CE is an extremely sensitive technique, making it ideal for the biological analysis of individual cells. However, a major limitation of single-cell CE is low throughput (5-35 cells per day). The aim of this project is to develop an automated single-cell CE platform that will dramatically improve throughput, enabling hundreds to thousands of cells to be analyzed per day. This automated CE instrument will then be used to measure SphK activity in LSCs from ML patients. This study will be the first to investigate a biological signalng pathway in human LSCs within individual patients. Characterizing SphK activity in LSCs is expected to contribute to the development of therapies that specifically target LSCs and therefore prevent AML relapse.